Abstract

During embryogenesis in mouse, the thymus is seeded by waves of hematopoietic stem cells
that provide the first peripheral T lymphocytes after birth. It is known that embryo thymocytes
and adult thymocytes have different phenotypic and functional features. The identification
of genes expressed in the thymus only during embryogenesis would help to understand
the molecular basis underlying these characteristics. We used the mRNA differential display
technique to compare gene expression between thymus and kidney from embryo (171/2 days)
and adult mice. This technique is the method of choice for comparing gene expression
because it is able to display rapidly and simultaneously the mRNA complement from several
different types of cells. The major drawback of the method is that it leads to the cloning of
many false positives and therefore needs a high throughput method to screen for the truly differentially
expressed cDNAs. We combined advantages from previously described methods
in order to develop a new version of the mRNA differential display technique that is fast,
cheap, and reliable. Instead of oligo dT priming, we used random hexameres for the reverse
transcription of total RNA and 10-mer primers for the amplification of internal parts of the
cDNAs. We obtained reproducible and clean patterns of discrete bands. We were able to easily
identify DNAs differentially amplified between embryo and adult tissues (embryo specific;
E 58.73), between thymus and kidney (thymus specific; Thy 52.54), or between embryo
and adult thymus (embryo thymus specific; E Thy 58.73) cDNA fragments. After reamplification,
cloning, and sequencing of these DNA fragments, it appeared that in most cases, one
band corresponded to a single DNA sequence. On a northern blot, each of these candidate
genes recognized a transcript that is differentially expressed as expected. Thus, we report an
optimized, reproducible, and fast mRNA differential display method that overcomes the usual
problems met with the originally described technique or its reported modifications.